IL-35 is a newly discovered cytokine that contributes to the suppressive capacity of Tregs. IL-35 Is a heterodimer consisting of an alpha (p35) and beta chain (Ebl3). It is a member of the IL-12 cytokine family, which is characterized by the sharing of 3 alpha (pi 9, p28, p35) and 2 beta (p40 and Ebi3) subunits, and also includes IL-12 (p35/p40), IL-23 (p19/p40) and IL-27 (p28/Ebi3). Additional information regarding the basis of subunit sharing, dimer formation, and other mechanisms to regulate IL-12 family members at the protein level will be critical for the development of IL-35-based therapeutics that do not interfere with function of IL-12 or IL-27. The objective of this research plan is to characterize the Interactions between IL-12 family subunits, particulariy IL-35 and the subunits that it shares with IL-12 and IL-27. This work will address the following questions: Alm#1: Is IL-35 secretion regulated at the protein level? My preliminary data indicate that IL-35 is poorly secreted by transfected cells, compared to IL-12 and IL-27, suggesting IL-35 formation is unfavorable. We will determine if activated dendritic cells, which express IL-12 and IL-27 are capable of secreting IL-35 by, IP/western blot. We will evaluate the ability of multiple cell types to ectopically express IL- 35, to determine if an unknown Treg-specific factor is required for optimal IL-35 production. We will assess relative chain pairing efficiencies within the IL-12 family by comparing intracellular and secreted cytokine levels in a normalized in vitro expression system by IP/western blot. Finally, we will monitor intracellular competition between IL-12 family subunits with a shared binding partner in FRET and BiFC assays. Aim#2: Do IL-35 subunits use the same binding interface for formation of IL-12 and IL-27. The development of theraputic applications of IL-35 that do not interfere with IL-12 and IL-27 will require extensive knowledge of the shared dimerization interfaces of each subunit. We will mutate p35 residues that are involved in formation of the IL-12 interface and assess their role in IL-35 formation using IP/western blot and functional assays. We will also mutate the putative binding interface of Ebi3 and determine if the same residues are involved in dimerization of both IL-27 and IL-35. Finally, we will attempt to identify residues in both p35 and Ebl3 that can abbrogate IL-35 formation and leave IL-12 and IL-27 function intact. Regulatory T cells play an important role in suppression of autoimmune diseases, and lack of Treg function can promote anti-tumor Immunity. To utilize Tregs to control disease, it is important to understand proteins that mediate their function. We will investigate the mechanisms that regulate expression of Treg- speclfic cytokine IL-35, a potential theraputic target for autoimmune diseases and cancer.